In the Laboratory
Synthesis and Separation of a Diastereomeric Sulfonium Ion by Capillary Zone Electrophoresis Francisco A. Valenzuela, Thomas K. Green, and Darwin B. Dahl* Department of Chemistry, Western Kentucky University, Bowling Green, KY 42101
Capillary electrophoresis (CE) is becoming increasingly popular as an analytical separation technique. Its attractive features include rapid separation of semivolatile and nonvolatile components; extremely high theoretical plate numbers; the ability to separate cationic, anionic, and neutral species; and an approximately 3-fold lower solvent demand than liquid chromatography (1). An additional advantage is that chiral separation is easily achieved by addition of a suitable chiral selector to the buffer. CE instrumentation is also becoming more prevalent in educational environments. The simplicity of instrumentation design and requirements has even enabled the building of homemade devices suitable for undergraduate laboratory experiments (2). The separation of enantiomers is an important area of analytical chemistry. Workers in pharmaceutical, agrochemical, and biological fields are especially interested in enantiomeric composition owing to the likelihood that different enantiomers will have different pharmacological effects (3). Because some isomers may cause undesirable side effects, the industry strives to prepare enantiomerically pure compounds. As a consequence, rapid and sensitive procedures are desired to provide quality control/assurance of certain optically active products. Historically, the large-scale separation of racemic mixtures was achieved by either fractional recrystallization of diastereomeric salts or microbiological digestion (4). No broad-scale technique offered a fast and efficient means for isolation. With the advent of chiral stationary phases, particularly in liquid chromatography, the number of published procedures has increased tremendously (5). Today, enantiomeric analysis is mainly achieved through liquid and gas chromatography and capillary electrophoresis, with CE gaining increasing attention (6 ). A literature survey of this Journal revealed four reported CE laboratory applications and only one of them involves the separation of optical isomers (2, 7–9). The widespread applications and projected utility of CE strongly argue for its introduction to the undergraduate student. Therefore, we present a laboratory exercise utilizing CE as the analytical tool in the analysis of a student-synthesized sulfonium ion, secbutylmethyl-p-tolylsulfonium tetrafluoroborate, which contains 2 stereogenic centers, thereby yielding four optical isomers. Sulfated cyclodextrin is used as the buffer modifier to achieve chiral separation.
Experimental Procedure
Instrumentation Separation was achieved using a BioRad BioFocus Model 3000 capillary electrophoresis system equipped with a 55.4-cm (working length) by 50-µm i.d. fused silica capillary column. Sample introduction was achieved by selecting the pressure mode set at 2 psi-sec. Operating potential was fixed at 17 kV (60 µA) normal mode and a column temperature of 15 °C. Detection wavelength was 264 nm ( λmax = 230 nm). Phosphate buffer used for the separations was modified by the addition of 50 mM tetrabutylammonium bromide and 4 mM sulfated cyclodextrin. Reagents and Materials Sodium phosphate buffer (0.10 M, pH 2.5) was purchased from BioRad. Tetrabutylammonium bromide (99%), sulfated β-cyclodextrin (degree of substitution, 7–11 moles of sulfate groups per mole of β-cyclodextrin), methyl-ptolylsulfide (99%), 2-iodobutane (99%), silver tetrafluoroborate (98%), and HPLC-grade water and acetonitrile were obtained from Aldrich. Dichloroethane (ACS reagent grade) was obtained from Aldrich and dried over 8–10 mesh, 4-Å molecular sieves (EM Science) before use. Synthesis of sec-Butylmethyl-p-tolylsulfonium Tetrafluoroborate The procedure of Acheson and Harrison was used for synthesis of the sulfonium salt (10). Approximately 1 mmol (0.138 g) of methyl-p-tolylsulfide and 1 mmol (0.184 g) of 2-iodobutane were dissolved in 2 mL of dry dichloroethane (DCE) in a 5-mL conical vial. A DCE solution of silver tetrafluoroborate (1.1 mmol of AgBF4 dissolved in 1 mL of DCE) is added to this stirred solution and allowed to react for 2 hours. The yellow precipitate (AgI) is centrifuged by a conventional bench-top centrifuge for 5 minutes and washed twice with 2 mL of acetonitrile. The supernatants are combined and the solvents are removed by rotovaporization at 40 °C to leave a colorless oil. The oil is dried under vacuum overnight at room temperature and used directly for CE and NMR analysis. CAUTION: Dichloroethane is a cancer suspect agent. Use caution in handling. Especially, make certain to use proper ventilation.
*Corresponding author.
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Journal of Chemical Education • Vol. 75 No. 12 December 1998 • JChemEd.chem.wisc.edu
In the Laboratory
Procedure A stock solution of the sulfonium salt is prepared by dissolving 15 mg in 1.0 mL of HPLC-grade acetonitrile. The working standard solution is prepared by adding 20 µL of the stock solution to 300 µL of pH 2.5 phosphate buffer and 200 µL of HPLC-grade water. The resulting concentration is approximately 2 mM. All solutions, buffer, and working standard are filtered through a 0.45- µm membrane filter (Gelman Sciences) before use. Separation buffer is placed in both anodic and cathodic 1.5-mL reservoirs. The capillary is preconditioned for 5 minutes with buffer before injection of the sample. Results and Discussion The synthesis of sec-butylmethyl-p-tolylsulfonium tetrafluoroborate involves a single-step process with nearly quantitative conversion. The product is easily isolated and its composition can be conveniently confirmed by NMR as an additional laboratory exercise if desired. Figure 1 shows the 270-MHz 1H NMR spectrum of the product in d6-acetone. Provided the students have an appropriate background, they can be asked to interpret this spectrum and to identify diastereomeric resonances. The S-methyl singlet is shown as two resonances at 3.5 ppm, each corresponding to a different diastereomer. The other methyl resonances are clearly distinguishable as two triplets near 1.0–1.2 ppm and two well-resolved doublets. Obviously, enantiomers cannot be distinguished in this spectrum, but the student is then asked to analyze the mixture using CE as a resolving technique. This presents a good opportunity for the instructor to discuss the inability of infrared and mass spectrometry to differentiate optical isomers. Figure 2 shows a representative electropherogram. The identification of each isomer is not reported owing to lack of appropriate standards. However, each isomer yields the same peak area, consistent with the formation of a racemic mixture of the sulfonium salt from the parent sulfide, as expected. Methyl-p-tolylsulfide is a possible impurity about which the initiated student may inquire in terms of potential interference. The migration time of this material is 36.7 minutes. The electroosmotic flow at a low pH of 2.5 is extremely slow and controls the migration rate of neutral compounds. Therefore, this starting material does not overlap with the sulfonium ion. The ease of the synthesis and speed of separation make this perfectly suited for a laboratory exercise involving several students. The total experiment requires approximately two 3-hour laboratory periods to complete. The first period is set aside for the synthetic work and the second for the separation. If time allows, student can explore parameters affecting the separation of the optical isomers. For example, by independently varying the column temperature, injection time, voltage, and cyclodextrin concentration, drastic changes in resolution can be seen and various conclusions can be generated. Our experience has shown that this experiment gives students a good appreciation of the concepts of diastereomers and enantiomers and operational concepts of CE. The added benefit of having students synthesize and analyze their own compound proves to be very satisfying to the students.
Figure 1. 1H NMR spectrum of sec -butylmethyl-p -tolylsulfonium tetrafluoroborate.
Figure 2. Electropherogram of four enantiomers/diastereomers of secbutylmethyl- p-tolylsulfonium tetrafluoroborate in a racemic mixture.
Acknowledgment We would like to acknowledge the NSF-ILI award DUE 9650340 for the acquisition of the CE system used in this work. Literature Cited 1. Li, S. F. Y. Capillary Electrophoresis—Principles, Practice and Applications; Elsevier: Amsterdam, 1992. 2. Conte, E. D.; Barry, E. F.; Rubinstein, H. J. Chem Educ. 1997, 73, 1169. 3. Ariens, E. J. Clin. Pharmacol. Ther. 1987, 42, 361. 4. Blaschke, G. Angew. Chem. Int. Ed. Engl. 1980, 19, 13. 5. Armstrong, D. W. CRC Critical Rev. Anal. Chem. 1988, 19, 175. 6. Novotny, M.; Soini, H.; Stefansson, M., Anal. Chem. 1994, 66, A632. 7. Thompson, L.; Veening, H.; Strein, T. G. J. Chem. Educ. 1997, 74, 1117. 8. Contradi, S.; Vogt, C.; Rohde, E. J. Chem. Educ. 1997, 74, 1122. 9. Weber, P. L.; Buck, D. R. J. Chem. Educ. 1994, 71, 609. 10. Acheson, R. M.; Harrison, D. R. J. Chem. Soc. C 1970, 13, 1764.
JChemEd.chem.wisc.edu • Vol. 75 No. 12 December 1998 • Journal of Chemical Education
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